The long-term goal is to understand the structural and functional mechanisms that control cytoskeleton-membrane dynamics in health and disease. Here, we will determine the structural and functional bases for the activities of two types of cytoskeleton- organelle scaffolds, the Arp2/3 complex activator WHAMM and the microtubule- organelle linker Hook. Aim 1 will test the hypothesis that WHAMM links actin polymerization through the Arp2/3 complex to organelle morphogenesis and transport during autophagy. WHAMM consists of an N-terminal membrane-binding domain of unknown structure and function and a C-terminal PWCA (Pro-rich, WH2, Central, Acidic) region responsible for actin polymerization by activation of Arp2/3 complex. Our initial studies support the hypothesis that WHAMM promotes actin polymerization to drive the segregation of membranous organelles from the ER during autophagosome formation. Bioinformatics analysis suggests that WHAMM contains a phosphoinositide-binding PX domain and a membrane-curvature sensing/inducing helical domain. To test these hypotheses, cellular studies will reveal the subcellular localization, function and binding partners of WHAMM and biochemical-structural studies will uncover the molecular architecture, lipid-binding activity, protein-protein and auto-inhibitory interactions that adapt WHAMM for its role in autophagy. Aim 2 will address the molecular mechanism for cargo tethering to the microtubule cytoskeleton by Hook isoforms. Three Hook isoforms function as specialized microtubule-cargo tethering molecules in mammalian cells. Hook contains an N-terminal microtubule-binding domain, followed by a coiled coil region and a variable C-terminal domain thought to mediate specific interactions with cargo molecules. Cellular studies have established at least 18 binding partners of Hook, with roles in processes such as endosomal sorting, receptor turnover, and centrosomal assembly. Yet, the molecular mechanism for Hook's function in cargo tethering is largely unknown. Our structural and biochemical studies will reveal the molecular bases for Hook interactions with microtubules and cargos, dimerization, autoinhibition and activation. Working with the other projects of this PPG, our approach is multi-disciplinary, spanning cell biology to atomic structure.